Production of mannosylated hrsACE2 by baculovirus expression system; the way to remove and redirect SARS-CoV-2 viral particles to macrophages

Abstract

Purpose The immune system combats viral infections through innate and adaptive responses, with macrophages and dendritic cells playing key roles in pathogen clearance. Neutralizing antibodies aid viral elimination but have variable efficacy against SARS-CoV-2, which uses ACE2 for entry. Human recombinant soluble ACE2 (hrsACE2) acts as a decoy to block viral entry but has limitations in affinity and immune uptake. We produced mannosylated hrsACE2 (Mann-hrsACE2) via a baculovirus expression system to enhance uptake by innate immune cells, aiming to improve viral neutralization and stimulate cell-mediated immunity against SARS-CoV-2.Methods Mann-hrsACE2 protein was produced in Sf9 insect cells using recombinant baculovirus, purified by Ni-NTA chromatography, and verified by SDS-PAGE and Western blot. Its interaction with macrophages was assessed by FITC-labeling and analyzed through fluorescence microscopy and flow cytometry.Results Expressed Mann-hrsACE2 displayed an upward shift SDS-PAGE and Western blot analysis. Flow cytometry revealed macrophage uptake of Mann-hrsACE2 at 3, 28, and 98 corresponding to concentrations of 1, 10, and 51 nM, respectively.Conclusion The increased molecular weight of the expressed protein confirmed its glycosylation. Its mannosylation enabled efficient uptake by macrophages, key antigen-presenting cells in the immune system. Furthermore, by binding to SARS-CoV-2, the protein not only inhibits viral entry but also redirects the virus to macrophages for enhanced immune clearance.